The substrate specificity of the human ATP binding cassette transporter ABCG2
Abstract
ATP Binding Cassette (ABC) transporters are present in a variety of organisms, and they play numerous important roles in biology. ABC transporters use the energy released from ATP hydrolysis to transport substrate molecules across cellular membranes. The substrates transported by ABC transporters are diverse, and the transport may serve different functions in the cell. Mutations and altered expression of ABC transporters have been implicated in many clinically relevant disorders. For instance, overexpression of the ABC transporter P-glycoprotein may result in multidrug resistant cells and multidrug resistance is a major obstacle for successful cancer chemotherapy. The ABC half-transporter ABCG2 can also confer drug resistance to cancer cells in vitro. ABCG2 and P-glycoprotein are capable of transporting many of the same substrates, although the substrate specificity of ABCG2 is distinct from that of P-glycoprotein. ABCG2 recognizes a wide variety of substrates, and the substrate specificity can be altered as a result of mutations. Whereas specific mutations in ABCG2 may abolish substrate transport altogether, others result in a protein with altered substrate recognition properties. In this thesis, work on the interaction of mutant and wild-type ABCG2 proteins with natural and endogenous substrates is presented. Specifically, I show that the activity of ABCG2 was not affected by the potent P-glycoprotein inhibitor cyclosporin A. In contrast, both ABCG2 and P-gp interacted specifically with the acetogenin mucocin. Porphyrins are important biomolecules and here I show that ABCG2 interacted specifically with protoporphyrin IX, a precursor in the heme biosynthesis pathway. Mutagenesis of arginine 482 of ABCG2 revealed that this residue is important for substrate translocation, but not for substrate binding. Using random mutagenesis in combination with transport assays several residues of the transmembrane domain of ABCG2 that are important for function were identified. Conclusively, the work in this thesis contributes to the understanding of how the ABC transporter ABCG2 interacts with and transports its substrates.
Degree
Ph.D.
Advisors
Hrycyna, Purdue University.
Subject Area
Molecular biology|Biochemistry
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